1. Field of the Invention
The present invention relates to a process for amplifying a specific segment of nucleic acid. Methods for amplifying nucleic acid can be used to facilitate the cloning of DNA and the characterization of both DNA and RNA sequences and so are useful methods for purposes of recombinant DNA technology and the study of molecular biology. In addition, the amplification of specific nucleic acid segments greatly facilitates the detection of pathogens and of disease states associated with the presence of particular nucleic acid sequences, so the present invention is also of importance in medical diagnostic procedures. The detection of specific nucleic acid sequences is also useful for purposes of forensic medicine and paternity and individuality determination.
2. Description of Related Disclosures
U.S. Pat. No. 4,683,202 discloses a process for amplifying a specific nucleic acid segment by the polymerase chain reaction (PCR). PCR involves the use of two oligonucleotide primers, an agent for polymerization, a target nucleic acid template, and successive cycles of denaturation of nucleic acid and annealing and extension of the primers to produce a large number of copies of a particular nucleic acid segment. The segment copied consists of a specific sequence of nucleotides from the target template. This specific sequence is defined by regions on the template that can hybridize to the primers and the nucleic acid sequence between those regions. PCR methodology has had a profound impact in the fields of molecular biology, recombinant DNA technology, forensic medicine, and medical diagnostic technology.
U.S. Pat. No. 4,683,195 discloses a variety of methods for using PCR to amplify and then detect or clone a given nucleic acid segment. European Patent Publication No. 237,362 further illustrates the utility of PCR in the detection of specific nucleic acid sequences by disclosing that PCR can be used in conjunction with labeled probes and "dot-blot" methodology to detect the presence of a nucleic acid sequence initially present in extraordinarily small amounts.
Methods for performing PCR are disclosed in Ser. No. 063,647, filed Jun. 17, 1987, which issued as U.S. Pat. No. 4,965,188, and which is a continuation-in-part (CIP) of Ser. No. 899,513, filed Aug. 22, 1986, now abandoned, which is a CIP of Ser. No. 828,144, filed Feb. 7, 1986, which issued as U.S. Pat. No. 4,683,195, and which is a CIP of Ser. No. 791,308, filed Oct. 25, 1985, which issued as U.S. Pat. No. 4,683,202, and which is a CIP of abandoned Ser. No. 716,975, filed Mar. 28, 1985, all of which are incorporated herein by reference.
European Patent Publication No. 258,017 discloses a thermostable DNA polymerase that significantly simplifies PCR methodology. PCR involves successive rounds of denaturation or "melting" of double-stranded nucleic acid. The denaturation process is often carried out by heating the sample of nucleic acid, although a nucleic acid helicase can also be used for this purpose. However, this heating process can destroy the activity of the agent for polymerization, typically a DNA polymerase, that is used in PCR. Consequently, DNA polymerase must be added to PCR reaction mixtures after each heat-denaturation step unless a thermostable polymerase is employed. European Patent Publication No. 258,017 discloses a number of such thermostable polymerases, of which the polymerase from Thermus aquaticus, Taq polymerase, is most preferred for use in PCR, because the enzyme can survive repeated cycles of heating and cooling in PCR.
Saiki et al., Jan. 19, 1988, Science 239:487-491, describe methods for using Taq polymerase in PCR. Saiki et al. also describe the amplification of a specific DNA segment present only once in a sample of 10.sup.5 to 10.sup.6 cells.
Methods for purifying Taq polymerase and for the recombinant expression of the enzyme are disclosed in Ser. No. 143,441, now abandoned, filed Jan. 12, 1988, which is a CIP of Ser. No. 063,509, filed Jun. 17, 1987, which issued as U.S. Pat. No. 4,889,818, which is a CIP Ser. No. 899,241, now abandoned, filed Aug. 22, 1986, each of which is incorporated herein by reference.
Instruments for performing automated PCR are disclosed in Ser. No. 899,061, filed Aug. 22, 1986, which is a CIP of Ser. No. 833,368, filed Feb. 25, 1986, now abandoned. Structure-independent amplification of DNA can also be useful in the DNA sequencing methods disclosed in Ser. No. 249,367, filed Sep. 23, 1988. The present invention can be applied in the generation of single-stranded DNA by the method termed "asymmetric PCR" disclosed in Ser. No. 248,896, filed Sep. 23, 1988. The disclosures of these related patents and applications are incorporated herein by reference.
PCR methodologies offer enormous practical advantages over any other known way to amplify nucleic acid sequences. On occasion, however, a given pair of PCR primers does not yield an amplification product, even though the target sequence is present in the reaction mixture. When faced with such a "no amplification" result, the practitioner must do extensive testing of reagents and operating techniques to determine the origin of the problem. Sometimes such testing reveals the cause of the problem to be the particular primers used in the amplification, because primers that hybridize to another region of the target sequence can provide efficient amplification. This problem of "no amplification" occurs infrequently but, prior to the present invention, unpredictably. The problem is compounded by the difficulty in determining whether the primers are the reason for the inefficient amplification results.
Scientists working in areas not involving the polymerase chain reaction have observed that certain nucleic acid sequences can form stable secondary structures, such as palindromic hairpin loops or compressed regions. Because the presence of such structures can lead to anomalous migration patterns during gel electrophoresis, i.e., as in DNA sequencing, researchers attempted to find means for preventing the formation of secondary structures in nucleic acids. Barr et al., 1986, Bio Techniques 4(5):528-532, reported that use of 7-deaza-2'-deoxyguanosine-5'-triphosphate (c.sup.7 dGTP) in dideoxy-sequencing reaction mixtures helped to to resolve abnormal and compressed regions in the sequencing gels.
The present invention results from a synthesis of these two arts and provides a method for structure-independent amplification of DNA by PCR.